The Effect of Endotoxin-Induced Inflammation on the Activity of the Somatotropic Axis in Sheep

The hypothalamic-pituitary-somatotropic (HPS) axis controls many physiological and pathophysiological processes. The phenomenon of insensitivity to growth hormone resistance (GHres) was previously reported to be due to the development of inflammation. Therefore, the primary aim of the study was to determine the impact of inflammation caused by lipopolysaccharides (LPS) on the secretory activity of the HPS axis in sheep. The further goal was to determine the effect of inflammatory factors on individual components involved in intracellular signal transduction to GH via the GH receptor (GHR). The research was carried out on 24 seasonal sheep kept under a short-day photoperiod, randomly divided into two groups. Before the experiment, the sheep estrous cycles were synchronized. The results of the current study in a sheep model showed that inflammation impairs the activity of the somatotropic axis. On the one hand, LPS injection stimulated (p < 0.01) GH secretion, and on the other hand, it reduced the liver's sensitivity to this hormone by directly reducing (p < 0.01) GHR expression and activating the GHR inhibitory signal transduction mechanism. A symptom of such an inhibitory postreceptor signaling pathway may be due to an increase in SOCS3 expression (p < 0.01). The effect of various inhibition pathways is a significant reduction in the expression of the main transcription activator IGF1-STAT5B (p < 0.05). The action of GHres in the liver resulted in the inhibition of IGF1 secretion, which in the long term may have negative consequences for growth and development. Our study suggests that disruption of the GH cell signaling pathway may be one of the important elements of the pathophysiology of inflammation. It can suppress growth and hepatic metabolism to spare energy expenditure.


Introduction
Te infammation disrupts the homeostasis of an organism and leads to endocrine system disorders [1][2][3].To induce systemic infammation, experimental animals are treated with a lipopolysaccharide (LPS), which is a part of the outer cell membrane of Gram-negative (−) bacteria.Te infammation mediators, which exhibit pleiotropic activity, are believed to play a crucial role in the communication between immunological and neuroendocrine systems [4].One of the endocrine axes, in which activity is modulated during infammation, is the hypothalamic-pituitary-somatotropic (HPS) axis.Resulting of the LPS treatment systemic infammation induces changes in the secretory activity of three major organs related to the HPS axis, namely, the hypothalamus, pituitary, and liver [1][2][3].Te hypothalamus is the main regulatory organ in this axis.Secreted by its nuclei neurohormones, growth hormone-releasing hormone (GHRH), which is secreted mainly in the arcuate nucleus (ARC), and somatostatin (SST), which is secreted mainly in the periventricular nucleus (PVN), are two main factors that afect the secretion of growth hormone (GH) in the anterior part of the pituitary (AP).Te GH secreted from the pituitary gland is bound by the growth hormone-binding protein (GHBP).Te complex thus created is fnally able to bind with the GH receptor (GHR) [5].GHR expression was observed in most tissues and organs, but it is mainly secreted in the liver, where its activation by the GH complex bound stimulates the insulin-like growth factor 1 (IGF1).It is believed that infammatory mediators directly infuence the synthesis and/or release of GH at the pituitary level, omitting the hypothalamic regulation of GH secretion.In addition, it has been observed that activation of the immune system inhibits the secretion of IGF1 [6].However, this efect seems to be attributed to the decrease in liver sensitivity to the GH action caused by infammation [7].It is worth noting that the increase in GH secretion is a common element of the pathophysiology of sepsis both in sheep and primates including humans [7,8], which suggests that sheep, as opposed to rodents, can be useful model animals in the studies considering the efects of interaction between the immune system and somatotropic axis.Terefore, the results of the studies on sheep can be valuable and useful for human medicine.
In some pathophysiological conditions, the mammalian organism was found to not respond to the action of GH commonly despite the high circulating level of this hormone.Tis specifc body reaction is known as growth hormone resistance (GHres).Te GHres symptoms seem to be homologous to those in the GH defciency.GH defciency may cause changes in the composition of the body, causing a reduction in lean body mass and an increase in fat mass [9], a reduction of the total amount of water in the organism [10], a decrease in bone density [11], a decrease of strength and endurance of muscles [12], harmful infuence on the cardiovascular system [13], decrease in resting energy expenditure [14], decrease in protein metabolism and synthesis [15], carbohydrate metabolism disorders [15], lipid and lipoprotein metabolism disorders [16,17], reduction of the total amount of collagen in the skin [17], changes in the organisms immune system response [15], and breeding disorders that manifest as a negative infuence on, e.g., ovarian follicle development and puberty [18].Causes of the GHres can be divided into the following two groups: inborn and acquired.Inborn GHres is, otherwise, known as Laron syndrome and it is caused by a genetic mutation of GHR.On the other hand, the acquired GHres was found to be caused by numerous factors such as inhibiting antibodies, malnutrition, diabetes, and renal or hepatic disorders [19].An increasing number of experimental data indicates that one of the factors that induce acquired GHres may be infammation.Most authors indicate the potential role of fbroblast growth factor 21 (FGF21), a protein that is as of yet poorly studied, in the induction of GHres.FGF21 is a protein hormone that regulates the adaptation of organisms to various conditions such as limited nutrients, cold, the amount of carbohydrates in diet, or stress, including immune stress.Tis factor can potentially infuence the target tissues through 4 fbrogenic growth factor receptors (FGFR1-4).FGF21 afects the entire organism and acts in an endo-or autocrine fashion, depending on the stimulus and the production sites [20].FGF21 is mainly synthesized in the liver [21] and adipocytes [22].It was found that in humans, the level of FGF21 is positively correlated with the Quetelet II indicator also known as the body mass index or BMI and GH level but negatively correlated with the IGF1 level [23].Moreover, its inhibiting infuence on the activity of the signal transducer and activator of transcription (STAT)5b in the liver [24] which with Janus kinase 2 (JAK2) is part of the main pathway through which GHR transduces the GH signal [25,26].Tese results suggest the potential role of FGF21 in GHres induction.Te stimulation of the immune system with LPS caused the development of the GHres as well [6].FGF21 can potentially mediate in the GHres mechanism, as its level increases after LPS administration, inhibiting the expression of GHR-STAT5B.Also, the infuence of LPS on JAK2 and STAT5B seems to be a confrmation of this thesis [27].Moreover, it was found that FGF21 may be involved in the modulation of the infammatory response due to its role in inhibiting the secretion of proinfammatory cytokines such as IL-1β, IL6, and TNFA [28].Te abovementioned facts appear to indicate that FGF21 induces GHres independently of proinfammatory cytokines and the only role of immunological stress is to activate FGF21.It is suggested that SIRT1 is involved in the regulation of FGF21 expression.However, this relationship was studied mainly in the context of metabolism regulation in the peripheral tissues such as the liver [29] or heart [30].Sirtuin 1 (SIRT1) is a member of the sirtuins family (SIRT1-7), which are highly conserved NAD + -dependent protein deacetylases and/or ADP ribosyltransferases [31].Sirtuins are considered to be one of the crucial regulators of a variety of cellular processes, including energy metabolism, stress response, tumorigenesis, and aging [32].In turn, in adipocytes, the SIRT1⟶FGF21 pathway seems to be reversed as Chau et al. observed that it was FGF21 that regulated SIRT1 expression [33].Also, the inhibiting infuence of SIRT1 on the GH-induced GHR phosphorylation of STAT5B at the level of the liver [34], which is the main mediator of growth hormone signal transduction among STAT proteins phosphorylated by GHR [35].Besides metabolism regulation, the role of SIRT1 was observed also in the modulation of the immune response.Liu et al. (2013) suggested that TLR4 signaling recruits SIRT1, which is then associated with the autoinhibitory mechanism of infammation [36].
Considering the unknown mechanism of infammationinduced resistance to growth hormone, the study's primary aim was to determine the infuence of the infammation on GH concentration and GH receptor expression, as well as the protein expression of GHR and IGF1 in the liver in the pathological state.Furthermore, genes' expression involved in the GH signaling pathway was determined on the level of the hypothalamus, anterior pituitary, and liver.Animals were euthanized 3 h or 9 h after i.v.injection of LPS, respectively.Tissues from the mediobasal hypothalamus (MBH) containing the arcuate nucleus (ARC), dorsomedial hypothalamus (DMH) containing periventricular nucleus (PVN), anterior pituitary (AP), and liver were collected and immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.Blood samples were collected every 15 minutes beginning 2 hours prior to the administration of LPS or saline, according to whether the group was LPS treated or served as the control.

Materials and Methods
Experimental procedures were approved (authorization no.WAW2/052/2018 from 23 March 2018) by the 2nd Local Ethics Committee of the Warsaw University of Life Sciences-SGGW (Warsaw, Poland).

qRT-PCR Assay.
Te gene expression analysis was performed according to the previously described protocol [37], with the use of NucleoSpin RNA/Protein kit (Macherey-Nagel, Dueren, Germany) to isolate the total RNA from collected tissues, Maxima ™ First Strand cDNA Synthesis Kit (Termo Fisher Scientifc, Waltham, MA, USA) for a reverse transcription, and FIREPol ® HOT EvaGreen qPCR Mix ® Plus kit (Solis Biodyne, Tartu, Estonia) for real-time PCR.Te primers used for each gene are listed in Table 1.PCR reactions were performed using a Rotor-Gene Q thermocycler (Qiagen, Germantown, MD, USA) with Rotor Gene Q software.Te NormFinder (Molecular Diagnostic Laboratory, Arhus University Hospital, Arhus, Denmark) was used to identify the optimal normalization gene, from among the selected genes: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone deacetylase 1 (HDAC1), beta-2-microglobulin (B2M), and cyclophilin C (PPIC).
Te results are presented in arbitrary units, calculated as the ratio of the target gene expression to the expression of the reference gene, with the appropriate control group normalized to a value of 1.

Radioimmunological Assay.
Te concentration of GH in plasma was determined using a double-antibody radioimmunoassay (RIA) method, employing antibovine GH and anti-rabbit c-globulin antisera, along with a bovine GH standard (NIDDK-GH-B-1003A).Te detailed characteristics of the antiserum and the assay method were thoroughly described by Dvorak et al. [42].Te assay's sensitivity for GH was 0.6 ng/mL, with intraassay and interassay coefcients of variation at 5.9% and 10.2%, respectively.
2.4.ELISA.Tissue samples were homogenized in 1 ml of ice-cold phosphate-bufered saline (pH 7.4) and then subjected to two freeze-thaw cycles to further disrupt the cell membranes.Ten, the samples were centrifuged for 5 min at 10000 × g and 4 °C.Te supernatants were collected and stored at −80 °C for further analysis.Te concentrations of GHR and IGF1, IGF1R, and STAT5B were determined using the following ELISA kits according to the manufacturer's instructions: Sheep GHR (growth hormone receptor) ELISA Kit, Sheep Insulin-like growth factor 1 ELISA Kit (ELK Biotechnology CO., Ltd., Denver, CO, USA).Total protein concentration was determined spectrophotometrically using the Bradford method and the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA).All absorbance measurements were performed on a SpectraMax iD3 microplate reader (Molecular Devices, San Jose, CA, USA).

Statistical Analysis.
Te statistical analysis was performed using TIBCO Statistica 13.3 (TIBCO Statistica Ltd., Palo Alto, CA, USA).Te signifcant diferences in proteins and gene expression between the experimental groups were determined using the Student's t-test to compare expression between the control and LPS-treated groups.Similarly, the concentration of GH in serum was determined using the Student`s t-test, and the last two measurements before slaughter were taken into account for the calculations.Te results are presented as the mean ± standard error of mean (SEM) and results p ≤ 0.05 were deemed statistically signifcant.

Efect of Peripheral LPS Injection on the Circulating
Concentration of GH.It was found that the plasma concentration of GH was increased (p < 0.05) in the LPS-treated groups both 3 and 9 h after the treatment in comparison to controls (Figure 1).

Efect of Peripheral LPS Injection on the Protein
Expression of GHR and IGF1 in the Liver.LPS treatment suppressed (p < 0.05) GHR protein expression in the liver both 3 and 9 h after the treatment compared to this receptor expression in the control animals (Figure 2; panel A).Moreover, the administration of LPS reduced (p < 0.05) the protein level of IGF1 in the liver 3 h after the treatment.On International Journal of Infammation the other hand, in the animals sacrifced 3h after the endotoxin administration the IGF1 expression in the liver was at the same level in comparison to the control group (Figure 2; panel B) 3.3.Hypothalamus.It was found that LPS treatment reduced (p < 0.05) GHRH gene expression in the MBH of ewes euthanized 9 h after the treatment.On the other hand, the expression of the gene encoding GHRH in the group sacrifced 3 h after the LPS treatment did not difer from this gene expression in the control group (Table 2).In the DMH, the administration of LPS caused an increase (p < 0.05) SST gene expression in the animals euthanized 3h after the treatment, while in the group sacrifced 9 h after the LPS injection, no changes in this gene expression were stated (Table 2).

Anterior Pituitary.
It was found that the expression of gene encoding GHRHR was increased (p < 0.05) in the AP collected 3 h after the LPS treatment, while no changes in this gene expression were stated in the AP collected 9 h after the LPS injection.It was also determined that the LPS 4 International Journal of Infammation injection decreased (p < 0.05) the expression of genes encoding SSTR1, SSTR2, SSTR3, and SSTR5 in the AP in animals euthanized 3 h and 9 h after the endotoxin administration.It was stated that the expression of gene encoding GH increased (p < 0.05) in the AP from ewes sacrifced 3 h after the LPS injection, on the other hand, this efect of endotoxin treatment was not observed in the glands collected 9 h after the injection (Table 3).
3.5.Liver.It was stated that the LPS-induced infammation suppressed (p < 0.05) expression of GHR gene in the liver collected 3 h and 9 h after the endotoxin administration in comparison to the control groups.Te expression of IGF1 mRNA was reduced (p < 0.05) by LPS treatment in the liver but only in tissues collected 9 h after the injection.It was also found that the expression of gene encoding STAT5B in the liver was decreased (p < 0.05) after the LPS treatment in all LPS-treated groups.Te gene expression of JAK2 was stimulated (p < 0.05) by the LPS injection but only in the liver dissected from 3 h after the treatment.Whereas the expression of suppressor of cytokine signaling 3 (SOCS3) mRNA was increased (p < 0.05) in both LPS-treated groups.
Endotoxin-induced infammation reduced (p < 0.05) SIRT1 mRNA expression in the liver, but only in those collected 3h after the treatment.Moreover, it was also found that the expression of gene encoding FGF21 was increased (p < 0.05) only in the livers collected 9 h after the injection of LPS (Table 4).

Discussion
Our study showed that the infammation caused by the LPS treatment stimulates the release of GH and the expression of its encoding gene in the AP in the 3 h after the injection.On  International Journal of Infammation the other hand, this stimulatory efect of acute LPS administration on the GH release seems to expire over time because in the 9 h after the LPS administration, this stimulatory efect on the GH gene expression has not been observed, while the circulating level of GH was still elevated.Te lack of parallelism in the changes in the blood  6 International Journal of Infammation concentration of GH and GH gene expression in the pituitary may result partly from the nature of this hormone's secretion.In the pituitary gland, GH is not released immediately after synthesis in the AP.Following synthesis by polysomes attached to the endoplasmic reticulum, the GH hormone is packaged in the Golgi apparatus, forming secretory granules that are then sent to the plasma membrane and stored until stimulation [43][44][45][46][47][48][49][50].Moreover, the reason for still elevated circulating GH concentration at 9 h after the LPS treatment may be an intense release of this hormone in the earlier period; however, it should be mentioned that GH is characterized by a relatively short half-life.In the organism, GH is cleared via the kidneys and/or GHR internalization and has a half-life of approximately 15-20 min [51].It is worth mentioning that the stimulatory efect of acute LPS administration on GH release has been previously reported both in sheep [52] and humans [53].In another study, acute endotoxin administration in rats decreased the concentration of circulating GH [54].However, a more recent study suggested a relationship between the dose of the toxin and the direction of its efect on GH secretion.It was shown that the LPS administrated at low doses stimulated GH release but at high doses, it suppressed this hormone secretion [55].Interestingly, our study on the sheep model suggests that the mechanisms leading to the infammatory-dependent stimulation of GH secretion are not entirely clear.Te secretion of GH in the AP is regulated by the hypothalamus and major regulatory factors include GHRH and SST [56,57].Our results showed that LPS did not increase GHRH gene expression in the MBH and even the level of GHRH mRNA was reduced 9 h after the endotoxin administration.Acute stress induced by LPS injection did not reduce SST mRNA expression in the DMH and even stimulated this gene expression in the 3 h after the treatment.Tese all suggest that in ewes, acute infammation did not stimulate GH secretion via modulation of its hypothalamic modulators.Te results of our study indicate that infammation induced by acute LPS injection modulates GH secretion acting primarily at the pituitary level.Tis efect could be included directly by the circulating endotoxin because our previous study showed the gene expression of TLR4 directly in the AP [58].Moreover, the in vitro study demonstrated that endotoxin directly stimulated GH release from cultured ovine pituitary cells [52].However, at least partially the infammatory-dependent changes in the GH secretion in the AP may be caused by blood-borne as well as locally synthetized infammatory cytokines.Our recent study showed that endotoxin injection induced timedependent changes in the gene expression of proinfammatory cytokines and their corresponding receptors [59].Tis study showed that at the early stage of infammatory response, the expression of IL1B, IL6, and TNFA was increased in the AP.Tis stimulatory efect of infammation on the gene expression of these cytokines gradually disappeared and at the end of the experiment, 9 hours after LPS injection, only an increase in the gene expression of IL6 was determined.Tese three cytokines can modulate GH secretion in the pituitary cells.Te role of IL1B in the direct regulation of GH secretion remains ambiguous.Te in vitro experiment on porcine pituitary cells showed that IL1B increased GH output but reduced the galanininduced GH secretion [60].Te study on rat anterior pituitary cells also showed stimulatory direct action of IL1B on GH secretion [61].On the other hand, the study on rat pituitary cells under serum-free conditions suggested a generally inhibitory action of IL1B on GH release [62].Te GH secretion in the AP could be modulated also by TNFA; in vitro experiments showed that this cytokine decreased GRH-stimulated GH release from cultured ovine pituitary cells [63].It is worth mentioning that our previous study showed that acute LPS injection-stimulated TNFA gene expression has the shortest reaction time among the studied cytokines [59].Te infammatory cytokine whose gene expression remained elevated in the AP throughout the entire experiment was IL6.Previous in vitro studies showed that IL6 stimulated GH synthesis and release [64,65].Te results of an in vitro study on pituitary cells from adult pigs showed that the role of IL6 in the modulation of GH secretion could be even more signifcant because it was found that IL6 not only stimulated GH release but also potentiated the efect of GH releasers [60].It is worth mentioning that the stimulatory efect of IL6 on the release of GH was also reported in the study on men infused with recombinant human (rh) IL6 via an antecubital vein.Tis showed that IL6 infusion led to a signifcant increase in GH, peaking 1 h after the beginning of the infusion [66].Moreover, research conducted on humans has shown a dose-dependent efect of the stimulatory infuence of IL6 on the GH secretion and the highest peak was reached when a dose of 3 μg/kg of body weight was used [67].On the other side, in the same study, no efect of TNFA on the basal secretion of GH was stated [67].However, TNFA and IL1B are recognized as the most potent inducers of IL6 encoding gene expression and it has been frmly established that nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) plays a pivotal role in orchestrating this regulatory process [68,69].Terefore, both IL1B and TNFA may also exert an indirect efect on GH through the stimulation of IL6.
Interestingly, although infammation had no stimulatory efect on the transcription of GHRH in the hypothalamus, in the pituitaries collected 3 h after LPS administration, an increased GHRHR mRNA level was stated.GHRHR plays a pivotal role in the regulation of GH synthesis and secretion because it is responsible for the signal transduction of the hypothalamic GHRH.GHRH stimulates GH secretion from somatotropic cells of the AP via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cyclic adenosine monophosphate (cAMP) production [70].Increased gene expression of GHRHR in the AP suggests increased sensitivity of this gland in the frst hours after LPS administration to GHRH stimulation, which at least partially might infuence on stimulation of GH secretion during acute immune/infammatory challenges.It is worth mentioning that a transient increase in the gene expression of GHRH in the AP may be caused by stress induced by infammation.It is well known that LPS injection activates the hypothalamicpituitary-adrenal axis leading to an increase in the blood International Journal of Infammation level of corticosteroids such as cortisol [71] and corticosterone [72].In vitro study on the rat somatotroph cell line, MtT/S showed that corticosterone stimulated the expression of GHRHR [73].Our results also showed that in most cases, the expression of SST receptors was reduced in the AP collected from endotoxin-treated ewes.SST is considered to be one of the main inhibitors of the HPS axis and a suppressor of GH secretion.It was previously reported that SST inhibited GH secretion by reducing intracellular cAMP and/ or hyperpolarizing the cells through SST receptors and it is involved in the transcriptional regulation of the GH gene [73].Terefore, infammatory-dependent changes in the pituitary expression of receptors for hypothalamic GHRH and SST may at least partially result in increased GH secretion.
Our study showed that systemic infammation caused by the LPS administration suppresses the IGF1 gene and protein expression in the liver in 9 h after the treatment.Interestingly, this increase was found despite the elevated circulating concentration of GH, which is considered to be a potent stimulator of IGF1 production [74,75].However, we found that the same as in the case of the AP, the liver endotoxin injection caused a signifcant reduction of GHR mRNA expression.Tis result is consistent with the results of a previous in vitro study which showed that LPS treatment through both MyD88-dependent and -independent TLR4 signaling pathways inhibited GHR promoter activity leading to the inhibition of GHR gene expression [76].Our results establish a novel cytokineindependent mechanism for a decrease in GHR expression in bacterial sepsis.Tis may suggest reduced expression of GHR in the liver which in turn causes decreased sensitivity of this organ to GH stimulation, a state that can be described as resistance to GH action.Tis may explain why increased GH release did not induce an increase in IGF1 secretion.However, it is worth pointing out that infammation may also cause disturbances in GHR signal transduction.It was found that endotoxin treatment decreased the expression of the gene encoding STAT5B and at the same time increased SOCS3 mRNA expression.Tis indicates that infammation induces a postreceptor inhibitory mechanism.It is well known that GH regulates IGF1 production through activation of STAT5B signaling cascade and that STAT5B is required for GH-induced IGF1 mRNA expression in the liver [77,78].Terefore, infammatory-dependent suppression of STAT5B expression in the liver may be another mechanism involved in the inhibition of IGF1 secretion.On the other hand, it was found that infammation increased gene expression of SOCS3 in the ovine liver, which also may profoundly negatively infuence the GHR-JAK2-STAT transduction pathway.SOCS3 exerts inhibitory control over the GHmediated JAK-STAT signaling pathway through various mechanisms.One mode of action involves SOCS3 competitively impeding the phosphorylation of STAT5B, a downstream efector in the JAK-STAT cascade.In addition, SOCS3 can directly bind to the GHR, hindering the recruitment and phosphorylation of STAT5B [79,80].Moreover, SOCS3's interaction with GHR initiates the formation of a complex, leading to the degradation of the GHR-JAK2 complex through processes like ubiquitination.Te presence of the SOCS box in SOCS3 facilitates the recruitment of Elongin BC, a complex involved in the ubiquitin-proteasome system.Tis interaction contributes to the ubiquitination and subsequent degradation of GHR and JAK2, culminating in the attenuation of JAK2 activity [81,82].Terefore, the infammatory-dependent inhibition of IGF1 secretion could result largely from increased SOCS3 expression.Increased expression of SOCS3 may also explain why the increase in the JAK2 gene expression determined in the livers collected 3 h after LPS injection did not infuence IGF1 production.Our study suggests that another inhibitor of GH signaling SIRT1 seems to be not involved in the suppression of GHR transduction during acute infammation.It was found that endotoxin injection even decreased the gene expression of SIRT1 in the livers of ewes 3 h after the treatment, whereas 9 h after the treatment SIRT1 mRNA expression did not difer from the control.It should be mentioned that SIRT1 due to its multidirectional inhibitory action directed at the GHR signal transduction is considered to be involved in the pathophysiology of GHres [26].Our results suggest that in the GHres induced by acute immune stress, the role of SIRT1 is marginal.In contrast, we found increased gene expression of FGF21 in the liver collected 9h after LPS injection.Tis allows us to assume that FGF21 could be also involved in the induction of GHres at the later stages of the infammatory response.FGF21 can act as an endocrine as well as a paracrine factor and is considered to be an important negative regulator of mammalian growth [83].It was found that in the liver, FGF21 lowers the concentrations of the active form of STAT5B, a major mediator of GH actions, and causes corresponding decreases in the expression of its target genes including IGF1.FGF21 also induces hepatic expression of IGF1 binding protein 1 and suppressor of cytokine signaling 2, which blunt GH signaling [24].It is worth mentioning that increased expression of FGF21 gene during endotoxin-induced infammation may result from its anti-infammatory properties which were reported in both in vitro and in vivo studies [84,85].It was found that its administration has a protective efect from the toxicity of LPS and sepsis [86].

Conclusions
Our study on the sheep model showed that infammation disturbs the activity of the somatotropic axis on the one hand stimulating the secretion of GH on the other hand reducing the sensitivity of the liver to this hormone action via direct reduction of GHR expression as well as by the activation of mechanism inhibiting the GHR signal transduction pathway.Te efect of GHres in the liver was suppressed IGF1 secretion which in the long term may have negative consequences for growth and development.It seems that infammation-induced resistance to GH may be one of the important elements through which infammation negatively afects the body's condition.Because the sheep is a recognized animal model in immunology and 8 International Journal of Infammation neuroendocrinology research, better understanding of the processes leading to the development of GHres as well as the consequences of GHres for growth and development may be valuable for human medicine.

Figure 2 :
Figure 2: Te efect of lipopolysaccharide treatment on the liver level of growth hormone receptor (GHR) (a) and insulin-like growth factor 1 (IGF1) (b) protein expression 3 and 9 hours (h) after the treatment.Statistically signifcant diferences were analyzed by the Student's t-test.Te results are presented in arbitrary units as the mean ± standard error of the mean (SEM) and results with p ≤ 0.05 were deemed statistically signifcant.Asterisk ( * ) indicates the statistically signifcant diferences.

Table 1 :
Primer descriptions.Te efect of lipopolysaccharide (LPS; 400 ng/kg; iv.) injection on the serum growth hormone (GH) concentration which was measured 3 and 9 hours (h) after the treatment.Signifcant diferences were analyzed by the Student's t-test.Te results are presented as the mean ± standard error of mean (SEM) and results p ≤ 0.05 were deemed statistically signifcant.Asterisk ( * ) shows the statistically signifcant diferences between controls and research groups.

Table 2 :
Te efect of lipopolysaccharide treatment on the growth hormone-releasing hormone (GHRH) and somatostatin (SST) genes in the mediobasal and dorsomedial hypothalamic nuclei.
−Te results are presented in arbitrary units as the mean ± standard error of the mean (SEM) and results with p ≤ 0.05 were deemed statistically signifcant.Arrows indicate signifcant diferences between the groups, while dashes indicate no efect.

Table 3 :
Te efect of lipopolysaccharide injection on the relative expression of the following genes: growth hormone-releasing hormone receptor (GHRH), somatostatin receptors (SSTR) 1-3 and 5, and growth hormone (GH) at the anterior pituitary level.
−Te results are presented in arbitrary units as the mean ± standard error of the mean (SEM) and results with p ≤ 0.05 were deemed statistically signifcant.Arrows indicate signifcant diferences between the groups, while dashes indicate no efect.

Table 4 :
Te efect of lipopolysaccharide injection on the relative expression of the following genes: growth hormone receptor (GHR), insulin-like growth factor 1 (IGF1), signal transducer and activator of transcription 5B (STAT5B), janus kinase 2 (JAK2), suppressor of cytokine signaling 3 (SOCS3), sirtuin 1 (SIRT1), and fbroblast growth factor 21 (FGF21) at the liver level.↑ Te results are presented in arbitrary units as the mean ± standard error of the mean (SEM) and results with p ≤ 0.05 were deemed statistically signifcant.Arrows indicate signifcant diferences between the groups, while dashes indicate no efect.